Resveratrol augments paclitaxel sensitivity by modulating miR-671-5p/STOML2/PINK1/Parkin-mediated autophagy signaling in A549 cell.

BACKGROUND: Paclitaxel (PTX) resistance has become a notable clinical concern of Non-small cell lung cancer (NSCLC). Our study aim is to investigate the effects of Resveratrol (RES) on NSCLC cells that have developed resistance to PTX. The NSCLC cell line A549 was employed in this investigation to establish a PTX-resistant NSCLC cell line, denoted as A549/PTX, and established tumor transplantaton model. The presence of miR-671-5p, Stomatin-like protein 2 (STOML2), and mitophagy biomarkers was evaluated using quantitative teal-time PCR (qRT-PCR) and western blot, The assessment of cell proliferation and apoptosis was conducted through the utilisation of colony formation and flow cytometry assays. The investigation of mitochondrial autolysosomes was conducted using transmission electron microscopy (TEM). Our results showed that the application of RES therapy resulted in a substantial improvement in the sansitivity of A549/PTX cells. RES exhibited an augmentation of apoptosis and a suppression of mitophagy in A549/PTX cells. RES induced an upregulation in the expression of miR-671-5p. This, in turn, leaded to the inhibition of STOML2, a protein that directly interacts with PINK1. In summary, our research indicates that RES improved the susceptibility of A549/PTX cells to PTX through miR-671-5p-mediated STOML2 inhibition.

Extracellular vesicle-transmitted miR-671-5p alleviates lung inflammation and injury by regulating the AAK1/NF-κB axis.

Mesenchymal stem cells regulate remote intercellular signaling communication via their secreted extracellular vesicles. Here, we report that menstrual blood-derived stem cells alleviate acute lung inflammation and injury via their extracellular vesicle-transmitted miR-671-5p. Disruption of this abundantly expressed miR-671-5p dramatically reduced the ameliorative effect of extracellular vesicles released by menstrual blood-derived stem cells on lipopolysaccharide (LPS)-induced pulmonary inflammatory injury. Mechanistically, miR-671-5p directly targets the kinase AAK1 for post-transcriptional degradation. AAK1 is found to positively regulate the activation of nuclear factor κB (NF-κB) signaling by controlling the stability of the inhibitory protein IκBα. This study identifies a potential molecular basis of how extracellular vesicles derived from mesenchymal stem cells improve pulmonary inflammatory injury and highlights the functional importance of the miR-671-5p/AAK1 axis in the progression of pulmonary inflammatory diseases. More importantly, this study provides a promising cell-based approach for the treatment of pulmonary inflammatory disorders through an extracellular vesicle-dependent pathway.

Circular RNA circARHGEF28 inhibited the progression of prostate cancer via the miR-671-5p/LGALS3BP/NF-κB axis.

Circular RNAs (circRNAs) play crucial roles in various biological processes, including prostate cancer (PCa). However, the precise roles and mechanism of circRNAs are complicated. Hence, we studied the function of a circRNA that might be involved in the progression of PCa. In this study, we found that circARHGEF28 was frequently downregulated in PCa tissues and cell lines. Furthermore, gain- and loss-of function experiments in vitro showed that circARHGEF28 inhibited proliferation, migration, and invasion of PCa. Additionally, circARHGEF28 suppressed PCa progression in vivo. Bioinformatics analysis and RNA pull-down and capture assay found that circARHGEF28 sponged miR-671-5p in PCa cells. Importantly, qRT-PCR and dual luciferase assays found that Lectin galactoside-binding soluble 3 binding protein (LGALS3BP) was downstream of miR-671-5p, and western blot analysis further confirmed that LGALS3BP negatively regulated the nuclear factor kappa-B (NF-κB) pathway. These results demonstrated that circARHGEF28 abolished the degradation of LGALS3BP by sponging miR-671-5p, thus blocking the activation of the NF-κB pathway. Our findings revealed that circARHGEF28/miR-671-5p/LGALS3BP/NF-κB may be an important axis that regulates PCa progression.

Silencing circ_0000644 inhibits papillary thyroid cancer cell malignancy by combining with miR-671-5p to release the inhibition on ANXA2.

BACKGROUND: Papillary thyroid cancer (PTC) is life-threatening due to its malignant progression. Considerable evidence demonstrates that circular RNA (circRNA) regulates PTC development. This study aims to explore the mechanism of circ_0000644 modulating PTC malignant progression. METHODS: The RNA levels of circ_0000644, microRNA-671-5p (miR-671-5p) and annexin A2 (ANXA2) were detected by quantitative real-time polymerase chain reaction. Western blot was performed to check protein expression. Cell proliferation and cell apoptosis were investigated by 5-ethynyl-29-deoxyuridine and flow cytometry. Angiogenic capacity, migration and invasion were analyzed by tube formation assay and transwell assay. The interaction between miR-671-5p and circ_0000644 or ANXA2 was identified by dual-luciferase reporter assay. Xenograft mouse model assay was performed to analyze the effect of circ_0000644 on tumor formation in vivo. RESULTS: Circ_0000644 and ANXA2 expression was significantly upregulated, while miR-671-5p was downregulated in PTC tissues and cells when compared with control groups. Circ_0000644 knockdown inhibited PTC cell proliferation, tube formation, migration, and invasion, but induced apoptosis in vitro. Moreover, circ_0000644 knockdown led to delayed tumorigenesis in vivo. In addition, circ_0000644 acted as a miR-671-5p sponge and mediated PTC cell tumor properties through miR-671-5p. ANXA2 was identified as a target gene of miR-671-5p, and its overexpression relieved miR-671-5p-induced effects in PTC cells. Furthermore, circ_0000644 depletion inhibited ANXA2 production by combining with miR-671-5p. CONCLUSION: Circ_0000644 depletion repressed PTC cell tumor properties through the miR-671-5p/ANXA2 axis.

Long non-coding RNA UCA1 regulates MPP+-induced neuronal damage through the miR-671-5p/KPNA4 pathway in SK-N-SH cells.

Parkinson's disease (PD) is an age-related neurodegenerative disease. Long non-coding RNA urothelial carcinoma-associated 1 (UCA1) is involved in the pathogenesis of PD. However, the pathogenesis of PD regulated by UCA1 has not been fully explained. We used 1-Methyl-4-phenylpyridinium (MPP+)-induced SK-N-SH cells for functional analysis. Expression levels of UCA1, microRNA (miR)-671-5p, and KPNA4 (karyopherin subunit alpha 4) mRNA were detected using quantitative real-time polymerase chain reaction (qRT-PCR). Cell viability and apoptosis were analyzed using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) or flow cytometry assays. Some protein levels were measured by western blotting. The levels of pro-inflammatory cytokines were tested by ELISA (enzyme-linked immunosorbent assay). The levels of LDH (lactate dehydrogenase), MDA (malondialdehyde), and SOD (superoxide dismutase) were measured using corresponding kits. The relationship between UCA1 or KPNA4 and miR-671-5p was verified by dual-luciferase reporter assay and/or RNA immunoprecipitation (RIP) assay. MPP+ induced UCA1 expression in SK-N-SH cells in a concentration-dependent manner or time-dependent manner. UCA1 knockdown reduced MPP+-induced apoptosis, inflammation, and oxidative stress in SK-N-SH cells. MiR-671-5p was downregulated while KPNA4 was upregulated in MPP+-treated SK-N-SH cells. UCA1 sponged miR-671-5p to regulate KPNA4 expression. MiR-671-5p inhibition counteracted UCA1 knockdown-mediated influence on apoptosis, inflammation, and oxidative stress of MPP+-induced SK-N-SH cells. KPNA4 overexpression offset the inhibitory influence of miR-671-5p mimic on apoptosis, inflammation, and oxidative stress of MPP+-treated SK-N-SH cells. UCA1 inhibition reduced MPP+-induced neuronal damage through the miR-671-5p/KPNA4 pathway in SK-N-SH cells, providing a novel mechanism to understand the pathogenesis of PD.

CircRNA-PTPRA Knockdown Inhibits Atherosclerosis Progression by Repressing ox-LDL-Induced Endothelial Cell Injury via Sponging of miR-671-5p.

Atherosclerosis (AS) is a chronic inflammatory disease with high morbidity and mortality rates worldwide. This study aimed to investigate the role of circular RNA protein tyrosine phosphatase receptor type A (circRNA_PTPRA) in oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cell (HUVECs) injury and its underlying molecular mechanism. The expression of circRNA-PTPRA and microRNA (miR)-671-5p was assessed by quantitative reverse transcription PCR (qRT-PCR). The interaction between circRNA-PTPRA and miR-671-5p was predicted using bioinformatic analysis. Cell viability and apoptosis were determined using the Cell Counting Kit-8 (CCK-8) assay and flow cytometry, respectively. Inflammation in HUVECs was analyzed by measuring the secretion of tumor necrosis factor alpha (TNF-α), interleukin-1beta (IL-1ß), and IL-6 using enzyme-linked immunosorbent assay (ELISA). Cleaved-caspase-3 expression was assessed using western blotting. The results indicated that circRNA-PTPRA expression was significantly increased and miR-671-5p expression was decreased in the serum of patients with AS and in ox-LDL-treated HUVECs. The interaction between circRNA-PTPRA and miR-671-5p was verified by dual luciferase reporter and RNA pull-down assays. In HUVECs, downregulation of circRNA-PTPRA reversed ox-LDL-induced reduction in cell viability, increase in apoptosis, and enhanced inflammation, whereas all these effects mediated by circRNA-PTPRA downregulation in ox-LDL-treated HUVECs were abolished by miR-671-5p downregulation. In conclusion, circRNA-PTPRA downregulation protects against ox-LDL-induced HUVECs injury by upregulating miR-671-5p, thereby providing potential therapeutic targets for AS.

Long Non-coding RNA ANRIL Downregulation Alleviates Neuroinflammation in an Ischemia Stroke Model via Modulation of the miR-671-5p/NF-κB Pathway.

The aim of this study was to investigate the role and underlying mechanism of the long non-coding RNA ANRIL (antisense noncoding RNA in the INK4 locus, ANRIL) in ischemia stroke (IS) injury. Downregulation of ANRIL by right intracerebroventricular injected si-ANRIL in middle cerebral artery occlusion-reperfusion (MCAO/R) C57/BL6 mice and by transferring si-ANRIL in oxygen glucose deprivation/reperfusion (OGD/R) HT22 cells. The results showed that ANRIL levels increased in IS model, downregulation of ANRIL reduced infract area, neurological deficit scores and injured cells, and prolong fall latency time in MCAO/R mice, improved cell viability and reduced cell cytotoxicity in OGD/R cells. Fluorescence in Situ Hybridization detected that there were both ANRIL and miR-671-5p in neurons; miranda v3.3a and dual luciferase reporter assay demonstrated that miR-671-5p was one of direct target of ANRIL; and our previously published research demonstrated that NF-κB was one of direct target of miR-671-5p. Downregulation of ANRIL alleviated neuroinflammation and reduced p-NF-κB, NF-κB, pro-inflammatory cytokines (IL-1ß, IL-6, TNF-a), and iNOS, which diminished by miR-671-5p antagomir both in in vivo and in vitro IS models. Downregulation of ANRIL alleviated disruption of blood brain barrier, and protected against tight junction (ZO-1, occludin and claudin 5) disorder in MCAO/R mice. This work clarified that downregulation of ANRIL reduced neuroinflammation by negatively regulating miR-671-5p to inhibit NF-κB in IS models, which provided a theoretical foundation for the protective effect of downregulating ANRIL for IS patients.

A novel LINC00943/miR-671-5p/ELAVL1 ceRNA crosstalk regulates MPP+ toxicity in SK-N-SH cells.

The competing endogenous RNA (ceRNA) activity of long non-coding RNAs (lncRNAs) has profound effects in pathological disorders, including Parkinson's disease. Here, we focused on the LINC00943-mediated ceRNA network for the regulation of LINC00943 in MPP+ toxicity in SK-N-SH cells. SK-N-SH cells were exposed to 1-methyl-4-phenylpyridinium (MPP+). LINC00943, miR-671-5p and ELAV like RNA binding protein 1 (ELAVL1) were quantified by real-time quantitative PCR (RT-qPCR) or western blot. Cell viability and apoptosis were gauged by Cell Counting Kit-8 (CCK-8) assay and flow cytometry, respectively. Direct relationship between miR-671-5p and LINC00943 or ELAVL1 was confirmed by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. Our data validated that LINC00943 regulated MPP+-evoked injury in SK-N-SH cells. LINC00943 regulated miR-671-5p expression by binding to miR-671-5p. Moreover, miR-671-5p was identified as a molecular mediator of LINC00943 in regulating SK-N-SH cell injury induced by MPP+. MiR-671-5p targeted and inhibited ELAVL1, and miR-671-5p-mediated inhibition of ELAVL1 impacted MPP+-evoked SK-N-SH cell injury. Furthermore, LINC00943 involved the post-transcriptional regulation of ELAVL1 through miR-671-5p competition. Our present study has established a novel mechanism, the LINC00943/miR-671-5p/ELAVL1 ceRNA crosstalk, for the regulation of LINC00943 on MPP+ toxicity in SK-N-SH cells.

Circ_0001360 absence alleviates oxygen-glucose deprivation/reoxygenation-induced SK-N-SH cell injury via controlling the miR-671-5p/BMF pathway.

BACKGROUND: The regulatory potency of circular RNA (circRNA) has been acknowledged in multiple human diseases, including ischaemic stroke (IS). However, only a few circRNAs were investigated in this disorder. We aimed to uncover the role of circ_0001360 in cell models of IS in vitro. METHODS: SK-N-SH cells were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) to simulate IS pathology conditions in vitro. Quantitative real-time PCR (qPCR) and western blot were applied for expression detection. Cell viability, proliferation and apoptosis were investigated by CCK-8, EdU and flow cytometry assays. The predicted binding of miR-671-5p to circ_0001360 or BMF 3'UTR was validated by dual-luciferase reporter and RIP assays. Proteins on the NF-κB pathway were quantified by western blot to assess NF-κB pathway activity. RESULTS: Circ_0001360 was upregulated in SK-N-SH cells after OGD/R treatment. OGD/R provoked SK-N-SH cell growth impairment, apoptosis and inflammation, while circ_0001360 knockdown relieved these injuries. Circ_0001360 targeted miR-671-5p, and miR-671-5p deficiency recovered SK-N-SH cell injury that was repressed by circ_0001360 knockdown. MiR-671-5p directly combined with BMF and repressed BMF expression. Accordingly, circ_0001360 targeted miR-671-5p to regulate the expression of BMF. Circ_0001360 knockdown weakened the phosphorylated levels of P65 and IκBα, while further miR-671-5p deficiency or BMF overexpression restored their expression levels. CONCLUSION: Circ_0001360 contributed to OGD/R-caused SK-N-SH cell injury via targeting the miR-671-5p/BMF network and activating the NF-κB pathway, thus participating in the development of IS.

miR-671-5p Attenuates Neuroinflammation via Suppressing NF-κB Expression in an Acute Ischemic Stroke Model.

This study was designed to investigate the role of miR-671-5p in in vitro and in vivo models of ischemic stroke (IS). Middle cerebral artery occlusion and reperfusion (MCAO/R) in C57BL/6 mice as well as oxygen-glucose deprivation and reoxygenation (OGD/R) in a mouse hippocampal HT22 neuron line were used as in vivo and in vitro models of IS injury, respectively. miR-671-5p agomir, miR-671-5p antagomir, pcDNA3.1-NF-κB, and negative controls were transfected into cells using riboFECT CP reagent. miR-671-5p agomir, pcDNA3.1-NF-κB, and negative vectors were administered into MCAO/R mice via intracerebroventricular injection. The results showed that miR-671-5p was significantly downregulated and that miR-671-5p agomir alleviated injury and neuroinflammation induced by ischemic reperfusion. A dual-luciferase reporter assay confirmed that NF-κB is a direct target of miR-671-5p. Reverse experiments showed that miR-671-5p agomir reduced neuroinflammation via suppression of NF-κB expression in both in vitro and in vivo models of IS. Our data suggest that miR-671-5p may be a viable therapeutic target for diminishing neuroinflammation in patients with IS.

Guizhi Fuling pills inhibit the proliferation, migration and invasion of human cutaneous malignant melanoma cells by regulating the molecular axis of LncRNA TPT1-AS1 / miR-671-5p.

The current research was aimed to explore the effects of Guizhi Fuling Pills on the proliferation, migration and invasion of human cutaneous malignant melanoma cells and its regulation on the molecular axis of LncRNA TPT1-AS1 / miR-671-5p. Human cutaneous malignant melanoma cells A375 were cultured in vitro and randomly divided into Con group, Guizhi Fuling pills-L group, Guizhi Fuling pills-M group, Guizhi Fuling pills-H group, Guizhi Fuling pills-H + pcDNA group, Guizhi Poria pills-H + pcDNA-TPT1-AS1 group. MTT was used to detect cell proliferation. The Transwell cell test was used to detect cell migration and invasion. qRT-PCR was used to detect the expression of TPT1-AS1 and miR-671-5p. The dual-luciferase report experiment verified the targeting relationship of TPT1-AS1, miR-671-5p. Western blot was used to detect the expression of Ki-67, PCNA, MMP-2 and MMP-9. Compared with Con group, Guizhi Fuling Pills could inhibit cell proliferation, migration and invasion (p<0.05), and also inhibit the expression of Ki-67, PCNA, MMP-2, MMP-9, TPT1-AS1 (p<0.05), promote the expression of miR-671-5p (p<0.05)., and the differences between the indexes of Guizhi Fuling Pill-L group, Guizhi Fuling Pill-M group and Guizhi Fuling Pill-H group were statistically significant (p<0.05). The dual-luciferase report experiment confirmed that TPT1-AS1 could target and bind to miR-671-5p and could regulate the expression and activity of miR-671-5p. Overexpression of TPT1-AS1 could reduce the inhibitory effect of Guizhi Fuling Pills on proliferation, migration and invasion of A375 cells. Guizhi Fuling Pill may reduce the proliferation, migration and invasion of human cutaneous malignant melanoma cells by down-regulating the expression of TPT1-AS1 and up-regulating the expression of miR-671-5p.

MiR-671-5p plays a promising role in restraining osteosarcoma cell characteristics through targeting TUFT1.

The aim of our study was to explore the roles of miR-671-5p in mediating biological processes of osteosarcoma (OS) cells and clinical implications. On the basis of the OS samples acquired from the GEO database, the expression difference and overall survival analyses of miR-671-5p and TUFT1 were determined. The expression of MiR-671-5p was verified using OS cell lines. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, wound-healing, and Transwell assays were respectively carried out to probe whether miR-671-5p regulated OS cell vitality, migration, and invasion. The expression of miR-671-5p was downregulated in OS tissues and cell lines. High expression of MiR-671-5p blocked OS cell growth, migration, and invasion. TUFT1 was predicted and validated as the target of miR-671-5p in OS cells using in silico analysis and luciferase reporter assays. Forced expression of TUFT1 reversed the suppressive influence of miR-671-5p on cell viability, migration, and invasion of OS cells. Moreover, the low expression of miR-671-5p and the high expression of TUFT1 led to poor prognosis. Taken together, targeting miR-671-5p/TUFT1 may be a promising strategy for treating OS.

Circular RNA circ_0001946 acts as a competing endogenous RNA to inhibit glioblastoma progression by modulating miR-671-5p and CDR1.

OBJECTIVES: In many malignant tumors, circRNAs play an important role. However, the biological role and clinical significance of circRNAs remain unclear. In this study, we investigated the effects of circ_0001946 on the progression of glioblastoma (GBM) and the molecular mechanism of circ_0001946. METHODS: Microarrays were applied to test the expression profiles of circRNAs and messenger RNAs (mRNAs). Coexpressed genes were identified by constructing differentially expressed circRNA-mRNA networks. The expression of circ_0001946, miR-671-5p, and cerebellar degeneration-related autoantigen 1 (CDR1) was detected by real-time quantitative PCR, and the protein expression of CDR1 was determined by western blotting. A dual-luciferase reporter assay was used to evaluate potential miR-671-5p target sites on circ_0001946 and CDR1. The proliferation, apoptosis, migration, and invasion of GBM cells were assessed by a colony formation assay, flow cytometry assay, transwell migration assay, and transwell invasion assay. Xenograft mouse models were used to determine the role of circ_0001946 in vivo. RESULTS: The expression of circ_0001946 and CDR1 was low and that of miR-671-5p was high in GBM cells. Circ_0001946 suppressed the expression of miR-671-5p, thus upregulating the expression of CDR1, the gene downstream of miR-671-5p. Circ_0001946 and CDR1 reduced proliferation, migration, and invasion and increased apoptosis in GBM cells, whereas miR-671-5p had an opposite effect. The xenograft mouse model and immunohistochemistry results indicated that circ_0001946 inhibited GBM growth as well as the expression of Ki67 in GBM cells. CONCLUSION: Our study confirmed that the circ_0001946/miR-671-5p/ CDR1 pathway modulates the development of GBM, and this pathway might be a promising target for the development of therapeutics for GBM.

Dynamically decreased miR-671-5p expression is associated with oncogenic transformation and radiochemoresistance in breast cancer.

BACKGROUND: Understanding the molecular alterations associated with breast cancer (BC) progression may lead to more effective strategies for both prevention and management. The current model of BC progression suggests a linear, multistep process from normal epithelial to atypical ductal hyperplasia (ADH), to ductal carcinoma in situ (DCIS), and then invasive ductal carcinoma (IDC). Up to 20% ADH and 40% DCIS lesions progress to invasive BC if left untreated. Deciphering the molecular mechanisms during BC progression is therefore crucial to prevent over- or under-treatment. Our previous work demonstrated that miR-671-5p serves as a tumor suppressor by targeting Forkhead box protein M1 (FOXM1)-mediated epithelial-to-mesenchymal transition (EMT) in BC. Here, we aim to explore the role of miR-671-5p in the progression of BC oncogenic transformation and treatment. METHODS: The 21T series cell lines, which were originally derived from the same patient with metastatic BC, including normal epithelia (H16N2), ADH (21PT), primary DCIS (21NT), and cells derived from pleural effusion of lung metastasis (21MT), and human BC specimens were used. Microdissection, miRNA transfection, dual-luciferase, radio- and chemosensitivity, and host-cell reactivation (HCR) assays were performed. RESULTS: Expression of miR-671-5p displays a gradual dynamic decrease from ADH, to DCIS, and to IDC. Interestingly, the decreased expression of miR-671-5p detected in ADH coexisted with advanced lesions, such as DCIS and/or IDC (cADH), but not in simple ADH (sADH). Ectopic transfection of miR-671-5p significantly inhibited cell proliferation in 21NT (DCIS) and 21MT (IDC), but not in H16N2 (normal) and 21PT (ADH) cell lines. At the same time, the effect exhibited in time- and dose-dependent manner. Interestingly, miR-671-5p significantly suppressed invasion in 21PT, 21NT, and 21MT cell lines. Furthermore, miR-671-5p suppressed FOXM1-mediated EMT in all 21T cell lines. In addition, miR-671-5p sensitizes these cell lines to UV and chemotherapeutic exposure by reducing the DNA repair capability. CONCLUSIONS: miR-671-5p displays a dynamic decrease expression during the oncogenic transition of BC by suppressing FOXM1-mediated EMT and DNA repair. Therefore, miR-671-5p may serve as a novel biomarker for early BC detection as well as a therapeutic target for BC management.

CircHDAC9 regulates myocardial ischemia-reperfusion injury via miR-671-5p/SOX4 signaling axis.

BACKGROUND: Myocardial ischemia-reperfusion (I/R), a harmful process in the treatment of cardiovascular diseases, can cause secondary damage to the cardiac tissues. Circular RNAs (circRNAs) are important regulators in a number of cardiac disorders. However, the role of circHDAC9 in myocardial I/R injury has not been clarified. METHODS: Human cardiac myocytes (HCMs) were treated with hypoxia/reoxygenation (H/R) and mice were subjected to I/R. Quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) was used to analyze the expression of circHDAC9, miR-671-5p, and SOX4, and western blot was used to detect SOX4 protein. The binding relationship among circHDAC9, miR-671-5p, and SOX4 was confirmed by RNA pull-down, luciferase, and RNA immunoprecipitation (RIP) assays. The effects of circHDAC9/miR-671-5p/SOX4 axis on the apoptosis, oxidative stress and inflammation were evaluated in both myocardial I/R injury models. RESULTS: The expression of circHDAC9 and SOX4 was noticeably elevated, whereas miR-671-5p expression was downregulated in both myocardial I/R injury models. circHDAC9 knockdown significantly reduced the apoptosis, activities of caspase-3 and caspase-9, ROS intensity, MDA activity, and concentrations of TNF-α, IL-1ß, and IL-6, but increased the viability and SOD activity in H/R-treated HCMs. Suppression of circHDAC9 dramatically reduced the levels of circHDAC9 and SOX4, while enhanced miR-671-5p expression in H/R-treated HCMs. CircHDAC9 functioned via sponging miR-671-5p to regulate SOX4 expression in vitro. Additionally, silencing of circHDAC9 improved the pathological abnormalities and cardiac dysfunction, and reduced the apoptosis, oxidative stress and inflammation in mice with myocardial I/R injury. CONCLUSIONS: Inhibition of circHDAC9 significantly improved myocardial I/R injury by regulating miR-671-5p/SOX4 signaling pathway.

MiR-671-5p sponging activity of circMMP1 promotes esophageal squamous cancer progression.

BACKGROUND: The aim of this study was to explore the function and mechanism of circular RNA (circRNA) matrix metallopeptidase 1 (circMMP1) in the progression of esophageal squamous cell carcinoma (ESCC). METHODS: CircMMP1 expression was detected by quantitative real-time PCR (qRT-PCR), and its relationship with the prognosis of ESCC patients was evaluated by Kaplan-Meier analysis. Cells were transfected using corresponding plasmids, and the cell proliferation activity, migration and invasion capabilities in vitro were assessed. The protein level in tissues and cells was analyzed using western blotting. RNA pulldown, dual-luciferase reporter assay and RNA immunoprecipitation assay were performed in ESCC cells to detect the interaction between circMMP1 and miR-671-5p, or the correlation between miR-671-5p and ANO1. Xenograft tumor experiment was carried out to uncover the function of circMMP1 in vivo. RESULTS: The high level of circMMP1 in tumor tissues was associated with poor prognoses of ESCC patients. Knockdown of circMMP1 suppressed ESCC cell proliferation, migration and invasion in vitro. MiR-671-5p was the target of circMMP1 and mediated the inhibition effect of circMMP1 on ESCC cells. CircMMP1 targeted miR-671-5p to regulate ANO1 expression, which was downstream of miR-671-5p. Overexpression of ANO1 weakened tumor-repressive function of circMMP1 knockdown in ESCC cells. Moreover, silencing of circMMP1 impeded ESCC tumor growth in vivo. CONCLUSION: Our study provided novel evidence that circMMP1 accelerated ESCC progression by acting as a miR-671-5p sponge to enhance ANO1 expression.

miR-671-5p Upregulation Attenuates Blood-Brain Barrier Disruption in the Ischemia Stroke Model Via the NF-кB/MMP-9 Signaling Pathway.

Blood-brain barrier (BBB) disruption can induce further hemorrhagic transformation in ischemic stroke (IS). miR-671-5p, a micro-RNA, is abundant in the cortex of mammalian brains. Herein, we investigated the roles and potential mechanisms for the effects of miR-671-5p on BBB permeability in IS. Results showed that miR-671-5p levels were significantly downregulated in the cerebral cortex of middle cerebral artery occlusion/reperfusion (MCAO/R) C57/BL6 mice in vivo. miR-671-5p agomir administration via right intracerebroventricular injection significantly reduced infarct volume, improved neurological deficits, the axon of neurons and nerve fiber, attenuated cell injury and apoptosis, as well as reduced BBB permeability in MCAO/R mice. Treatment with miR-671-5p agomir alleviated tight junction proteins degradation, including claudin, occludin, and ZO-1 in MCAO/R mice, and these effects were reversed following NF-κB overexpression. Bend.3 brain endothelial cells were subjected to oxygen and glucose deprivation/reoxygenation (OGD/R) treatment in vivo, and then miR-671-5p agomir was transfected into the cells. This resulted in reduction of cytotoxicity, improved cell viability, trans-endothelial electrical resistance, reduced fluorescein sodium permeability, and inhibited tight junction degradation in Bend.3 OGD/R cells. However, these effects were reversed following NF-κB overexpression. These results demonstrated that upregulation of miR-671-5p in IS models in vivo and in vitro alleviated BBB permeability by targeting NF-κB/MMP-9. In summary, miR-671-5p is a potential therapeutic target for protecting BBB permeability in IS to minimize cerebral hemorrhage transformation.

Circular RNA circSMAD4 regulates cardiac fibrosis by targeting miR-671-5p and FGFR2 in cardiac fibroblasts.

Heart failure is a leading cause of death and is often accompanied by activation of quiescent cardiac myofibroblasts, which results in cardiac fibrosis. In this study, we aimed to identify novel circular RNAs that regulate cardiac fibrosis. We applied transverse aortic constriction (TAC) for 1, 4, and 8 weeks in mice. RNA sequencing datasets were obtained from cardiac fibroblasts isolated by use of a Langendorff apparatus and then further processed by use of selection criteria such as differential expression and conservation in species. CircSMAD4 was upregulated by TAC in mice or by transforming growth factor (TGF)-ß1 in primarily cultured human cardiac fibroblasts. Delivery of si-circSMAD4 attenuated myofibroblast activation and cardiac fibrosis in mice treated with isoproterenol (ISP). si-circSmad4 significantly reduced cardiac fibrosis and remodeling at 8 weeks. Mechanistically, circSMAD4 acted as a sponge against the microRNA miR-671-5p in a sequence-specific manner. miR-671-5p was downregulated during myofibroblast activation and its mimic form attenuated cardiac fibrosis. miR-671-5p mimic destabilized fibroblast growth factor receptor 2 (FGFR2) mRNA in a sequence-specific manner and interfered with the fibrotic action of FGFR2. The circSMAD4-miR-671-5p-FGFR2 pathway is involved in the differentiation of cardiac myofibroblasts and thereby the development of cardiac fibrosis.

Circ_0001947 promotes cell proliferation, invasion, migration and inflammation and inhibits apoptosis in human rheumatoid arthritis fibroblast-like synoviocytes through miR-671-5p/STAT3 axis.

BACKGROUND: Circular RNAs (circRNAs) have emerged as vital regulators in the development of rheumatoid arthritis (RA). In this study, we aimed to explore the functions and mechanisms of circ_0001947 in RA. METHODS: The expression of circ_0001947, microRNA-671-5p (miR-671-5p) and signal transducer and activator of transcription 3 (STAT3) was determined by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot. Cell Counting Kit-8 (CCK-8) assay, 5'-ethynyl-2'-deoxyuridine (EdU) assay, flow cytometry analysis, transwell assay and wound-healing assay were performed to assess cell proliferation, apoptosis, invasion and migration. The concentrations of inflammatory factors were examined with enzyme-linked immunosorbent assay (ELISA) kits. Dual-luciferase reporter assay was used to analyze the relationships of circ_0001947, miR-671-5p and STAT3. RESULTS: Circ_0001947 was upregulated in RA patients and RA-FLSs. Knockdown of circ_0001947 repressed cell proliferation, invasion, migration and inflammatory response and facilitated apoptosis in RA-FLSs. Circ_0001947 served as the sponge for miR-671-5p and the inhibitory effect of circ_0001947 in RA-FLS progression was reversed by miR-671-5p inhibition. STAT3 was the target gene of miR-671-5p. MiR-671-5p overexpression restrained RA-FLS growth, invasion, migration and inflammation and promoted apoptosis, but STAT3 upregulation reversed the impacts. CONCLUSION: Circ_0001947 contributed to the progression of RA-FLSs by elevating STAT3 through adsorbing miR-671-5p.

Exosomal circ_PTPRA inhibits tumorigenesis and promotes radiosensitivity in colorectal cancer by enriching the level of SMAD4 via competitively binding to miR-671-5p.

Accumulating evidence supports that exosomal RNAs are crucial in tumor microenvironment and may be used as diagnostic biomarkers for cancers. This study aimed to determine the role of exosomal circular RNA_protein tyrosine phosphatase receptor type A (circ_PTPRA) in colorectal cancer (CRC). The morphology of exosomes was identified by transmission electron microscopy (TEM), and several exosome-specific proteins were quantified by western blot. The expression of circ_PTPRA, miR-671-5p and SMAD family member 4 (SMAD4) was detected using quantitative polymerase chain reaction (qPCR). Cell cycle was assessed using flow cytometry assay. Cell proliferation was assessed by MTT assay. Radiosensitivity was observed according to colony growth and cell apoptosis rate by colony formation assay and flow cytometry assay. The protein levels of proliferation- and apoptosis-related markers and SMAD4 were measured by western blot. The predicted relationship between miR-671-5p and circ_PTPRA or SMAD4 was verified by dual-luciferase reporter assay. Animal study was performed to investigate the role of exosomal circ_PTPRA in vivo. Circ_PTPRA expression was declined in serumal exosomes from CRC patients and CRC cell lines. Exosomal circ_PTPRA induced CRC cell cycle arrest and inhibited cell proliferation. Besides, exosomal circ_PTPRA promoted radiosensitivity of CRC cells, leading to inhibitory colony formation and increased apoptotic rate. In mechanism, circ_PTPRA functioned as a competing endogenous RNA (ceRNA) to increasing SMAD4 level by binding to miR-671-5p. Rescue experiments concluded that circ_PTPRA inhibited CRC growth and radioresistance by decreasing miR-671-5p expression, and miR-671-5p inhibition also inhibited CRC growth and radioresistance by enriching SMAD4 expression. Moreover, exosomal circ_PTPRA blocked tumor growth in vivo. Exosomal circ_PTPRA enhanced CRC cell radiosensitivity and inhibited CRC malignant development partially by regulating the miR-671-5p/SMAD4 pathway, hinting that exosomal circ_PTPRA might be used as a potential predicted and therapeutic target for CRC.